Platform For Immunotherapy Research Articles

EXTH-71. CHEMOTACTIC RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV) VIROTHERAPY IMPROVES LYMPHOCYTE RECRUITMENT IN GLIOBLASTOMA (GBM)

Abstract BACKGROUND Although immunotherapy holds promise for producing durable outcomes in GBM, a major obstacle is the limited ability of cytotoxic T lymphocytes (CTLs) to home to the tumor. CTLs are recruited via long-range signaling mediated by the diffusion of chemokines present in inflammatory environments. During glioma formation, tumors manufacture immune-suppressive chemokines and cytokines that co-opt resident cells, resulting in the preferential recruitment of immune suppressor cells from the periphery. We explored a novel strategy to selectively reprogram the tumor microenvironment (TME) using focal delivery of rAAV encoding a powerful call-and-receive chemokine for CTLs: CXCL9. HYPOTHESIS: rAAV transduction of the GBM TME to express CXCL9 (rAAV6-CXCL9) will enhance CTL tumor infiltration and improve responsiveness to immunotherapy. METHODS Proteomic arrays were used to identify CTL chemokines absent in human glioma. 3D immunohistochemistry and flow cytometry was used to evaluate geospatial rAAV transgene expression and lymphocyte trafficking in vitro and in vivo. Survival studies of rAAV6-CXCL9 alone and in combination with anti-PD-1 checkpoint blockade was performed in preclinical models of GBM. Tumor immunogenicity following treatment was evaluated by scRNAseq and proteomic analysis. RESULTS We identify rAAV6 as a reliable capsid for transducing murine tumor-reactive astrocytes. Transgene expression following a single intra-tumor injection is focal, stable, and results in high levels of CXCL9 production. CXCL9 is effective at promoting tumor T-cell chemotaxis in vitro and in vivo. rAAV6-CXCL9 treatment sensitizes GBM to PD-1 blockade, improving survival in two distinct syngeneic preclinical models- an effect that is largely dependent on CD8 T-cells. CONCLUSIONS We combine the excellent safety profile of rAAV with focal delivery, enhancing exposure within the tumor compartment and mitigating systemic toxicities seen with conventional therapies. rAAV therapy was successful at stimulating the intended biological response to our encoded transgene (CXCL9): increased lymphocyte recruitment. This strategy has far-reaching potential across other immunotherapy platforms.

STEM-23. SLOW CYCLING CELL RNA BASED T CELL THERAPY TO PREVENT RECURRENCE IN GBM

Abstract Glioblastoma (GBM), the most common primary malignancy of the central nervous system (CNS), is almost universally fatal due to inevitable recurrence despite aggressive therapeutics. Considering the infiltrative nature of GBM, targeting residual cells after surgical tumor resection is challenging. Development of effective novel adjuvant therapies against drivers of disease recurrence is of imperative priority. Anticancer treatments most effectively eliminate rapidly dividing cells but spare dormant or infrequently dividing populations. Multiple studies have reported in numerous cancers the existence of slow-cycling cells (SCCs) that are refractory to therapies. Our laboratory identified SCCs in high-grade glioma, which represents an enriched reservoir of highly infiltrative and treatment-resistant cancer propagating cells known to drive recurrence. An effective approach to specifically eliminate this cell population is urgently required to limit recurrence and improve survival. Importantly, these cells reliably defined a cellular niche characterized by definite potentially immunogenic neoantigens, which can serve as immune targets. Our ultimate goal is to leverage the power of adoptive cellular therapy (ACT) to recognize GBM SCC-derived antigens to achieve robust tumor control through targeting of these critical drivers of recurrence. Our data revealed that dendritic cells electroporated with slow-cycling cell RNA successfully primed T cells to recognize and target glioma treatment resistant cells. These experiments demonstrated that slow cycling cell-specific T cells (SCC-T cells) exhibit enhanced activation when in presence of treatment-resistant tumor cells as shown by increased CD8 differentiation, amplified effector and memory differentiation compared to total tumor RNA-specific T cell and control groups. Additionally, SCC-T cells presented the highest anti-tumor activity, as demonstrated by the greatest decrease of tumor cell proliferation and increase of T cell apoptosis. Together these results demonstrate the efficacy and superiority of SCC-based immunotherapy platform to target treatment-resistant glioblastoma cells.

Benefits of an Immunogenic Personalized Neoantigen Nanovaccine in Patients with High-Risk Gastric/Gastroesophageal Junction Cancer.

Personalized neoantigen vaccines have shown strong immunogenicity in clinical trial, but still face various challenges in facilitating an efficient antitumor immune response. Here, a personalized neoantigen nanovaccine (PNVAC) platform for adjuvant cancer immunotherapy is generated. PNVAC triggers superior protective efficacy against tumor recurrence and promotes longer survival than free neoantigens, especially when combined with anti-PD-1 treatment in a murine tumor model. A phase I clinical trial (ChiCTR1800017319) is initiated to evaluate the safety, immunogenicity, and prophylactic effect of PNVAC on preventing tumor recurrence in patients with high-risk gastric/gastroesophageal junction cancer after adjuvant chemotherapy of postsurgical resection. The one- and two-year disease-free survival rates are significantly higher than historical record. PNVAC induces both CD4+ and CD8+ T cell responses as well as antigen-experienced memory T cell phenotype. Furthermore, the immune response is persistent and remains evident one year after the vaccination. This work provides a safe and feasible strategy for developing neoantigen vaccines to delay gastric cancer recurrence after surgery.

Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.

Sulfonium-Driven Neoantigen-Released DNA Nanodevice as a Precise Vaccine for Tumor Immunotherapy and Prevention.

Peptide-based neoantigen vaccines hold tremendous potential for personalized tumor immunotherapy. However, effective delivery and controllable release of antigen peptides remain major challenges in stimulating robust and sustained immune responses. Programmable DNA nanodevices provide accurate fixed positions for antigens, which are convenient for the calculation of clinical dosage, and hold great potential as precise carriers. Here, a peptide-nucleic acid conjugate was prepared, which was driven by a propargyl sulfonium-based efficient and reversible bio-orthogonal reaction under weakly alkaline conditions, and folded into regular DNA nanodevice vaccines. The well-defined nanoplatform not only exhibits outstanding stability in serum, satisfactory safety, and effective internalization by antigen-presenting cells (RAW264.7 and BMDCs) but also obviously enhances cytokine (TNF-α, IL-6, and IL-12) secretion for further immune response. In vivo, the nanovaccine cooperating with OVA model antigens and CpG adjuvants stimulated an antigen-specific CD8+T cell response, significantly preventing the lung metastases of melanoma. In the B16-OVA tumor-bearing model, the growth inhibition rate of melanoma reached up to 50%. Similarly, the DNA nanodevice with neoantigen induced up to a maximum degree of complete MC-38 tumor regression in 80% of mice, possibly owing to antigen peptide reversible release driven by sulfonium and further cross-presentation. In brief, this study demonstrates that DNA nanodevices with sulfonium centers can provide a precise, biocompatible, and effective co-delivery vaccine platform for tumor immunotherapy and prevention.

Injectable Immunotherapeutic Hydrogel Containing RNA-Loaded Lipid Nanoparticles Reshapes Tumor Microenvironment for Pancreatic Cancer Therapy.

Pancreatic cancer immunotherapy is becoming a promising strategy for improving the survival rate of postsurgical patients. However, the low response rate to immunotherapy suggests a low number of antigen-specific T cells and a high number of immunosuppressive tumor-associated macrophages in the pancreatic tumor microenvironment. Herein, we developed an in situ injectable thermosensitive chitosan hydrogel loaded with lipid-immune regulatory factor 5 (IRF5) mRNA/C-C chemokine ligand 5 (CCL5) siRNA (LPR) nanoparticle complexes (LPR@CHG) that reprogram the antitumoral immune niche. The LPR@CHG hydrogel upregulates IRF5 and downregulates CCL5 secretion, which contribute to a significant increase in M1 phenotype macrophages. Tumor growth is controlled by effective M1 phenotype macrophage that initiate T cell-mediated immune responses. Overall, the LPR@CHG hydrogel is expected to be a meaningful immunotherapy platform that can reshape the immunosuppressive tumor microenvironment and improve the efficacy of current pancreatic immunotherapies while minimizing systemic toxicity.

Hybrid M13 bacteriophage-based vaccine platform for personalized cancer immunotherapy

Although cancer vaccines exhibit great advances in the field of immunotherapy, developing an efficient vaccine platform for personalized tumor immunotherapy is still a major challenge. Here we demonstrate that a bioactive vaccine platform (HMP@Ag) fabricated with hybrid M13 phage and personal tumor antigens can facilitate delivery of antigens into lymph nodes and activate antigen-presenting cells (APCs) through the Toll-like receptor 9 (TLR9) signaling pathway, which boosts both innate and adaptive immune response. As an adjuvant platform, hybrid M13 phages can deliver various tumor-specific antigens through simple adsorption to support the current development of personalized vaccines for cancers. Notably, the HMP@Ag vaccine not only prevented the tumors, but also delayed the tumor growth in established (subcutaneous and orthotopic) and metastatic tumor-bearing models while synergy with immune checkpoint blockade (ICB) therapy. Moreover, HMP@Ag triggered a robust neoantigen-based specific immune response in tumor-specific mutation models. In a clinically relevant surgery model, using autologous cell membrane from primary tumors-based HMP@Ag cooperation with ICB dramatically inhibited the post-operation recurrence, and elicited a long-term immune memory effect simultaneously. These findings imply that the M13 phage represents a powerful tool to develop a bio-activated hybrid platform for personalized therapy.

635 Reprogramming the tumor microenvironment by a second-generation recombinant modified vaccinia virus Ankara

Immune checkpoint blockade (ICB) therapy has brought hope to many cancer patients, but the response rate is low in many cancer types, and acquired resistance to ICB can develop over time. Oncolytic viruses are promising therapeutic agents for advanced cancers. Modified vaccinia virus Ankara (MVA) is an attenuated, replication-deficient poxvirus safe for human use, making it a favorable platform for cancer immunotherapy. Our first-generation recombinant MVA has shown promising antitumor efficacy in multiple murine tumor models due to the deletion of the E5R gene (encoding an inhibitor of the DNA sensor cGAS) from the MVA genome and the insertion of two membrane-anchored transgenes – Flt3L and OX40L, which leads to the activation of the host innate and adaptive antitumor immunity.

A prodrug hydrogel with tumor microenvironment and near-infrared light dual-responsive action for synergistic cancer immunotherapy

Immunotherapy has been used for cancer treatment, while it faces the common dilemmas of low therapeutic efficacy and serious immunotoxicity. In this study, we report the construction of a tumor microenvironment and near-infrared (NIR) light dual-responsive prodrug hydrogel for cancer synergistic immunotherapy in a more effective and safe manner. Such prodrug hydrogels were in-situ formed via calcium-induced gelation of alginate solution containing protoporphyrin IX (PpIX)‐modified iron oxide (Fe3O4) nanoparticles and programmed death ligand 1 antibody (aPD-L1) prodrug nanoparticles crosslinked by reactive oxygen species (ROS)-responsive linkers. PpIX served as a photosensitizer to produce singlet oxygen (1O2) under NIR laser irradiation for photodynamic therapy (PDT), and Fe3O4 nanoparticles mediated chemodynamic therapy (CDT) to generate hydroxyl radical (·OH) via Fenton reaction in the tumor microenvironment. In view of the cumulative actions of PDT and CDT, amplified ROS was generated to not only induce immunogenic cell death (ICD), but also destroy ROS-responsive linkers to achieve on-demand release of aPD-L1 from prodrug nanoparticles. Boosted antitumor immunity was elicited in tumor-bearing mice due to the aPD-L1-mediated immune checkpoint blocking. As a result, the prodrug hydrogel-based synergistic immunotherapy could almost treat bilateral tumors and prevent lung and liver metastasis using 4T1 tumor mouse models. This study thus offers a dual-responsive prodrug hydrogel platform for precision cancer immunotherapy. Statement of significanceVia calcium-induced gelation of alginate, we constructed a prodrug hydrogel with tumor microenvironment and near-infrared light dual-responsive action for synergistic cancer immunotherapy. Such hydrogels can achieve on-demand release of aPD-L1 upon photoactivation in the tumor microenvironment. Through mediating photodynamic and chemodynamic therapy, the prodrug hydrogels can induce enhanced immunogenic cell death and synergistically improve the efficacy of aPD-L1-mediated immune checkpoint blocking. The prodrug hydrogel-based synergistic therapy almost deracinates the primary and distant tumors, and prevents lung and liver metastasis in tumor mouse models.

Abstract 4157: Using a multivalent immunotherapy platform to extend intratumoral therapeutic durability

Abstract Purpose: There is a critical need in immunotherapy drug development to enable focused and sustained immune cell modulation within a tumor to induce and propagate a system-wide anti-tumor response. We have developed a novel immunotherapy platform that could be used to generate geographically focused cancer cell growth inhibition or immune cell activation, thereby stimulating an anti-tumor immune response against primary solid tumors that can also travel to secondary metastases. Methods: Using published methods, we synthesized multivalent protein (MVP) conjugates by conjugating multiple copies (i.e. valency) of immune stimulating proteins, checkpoint inhibitors or anti-tumor antibodies to soluble, long-chain biopolymers. We verified that we can reproducibly generate MVP valencies ranging from 20-120 protein copies (±10%) per polymer backbone. We determined the binding affinity of these MVPs to their respective targets using biolayer interferometry and cell bioassays, and we measured the hydrodynamic radius of these immunotherapies using dynamic light scattering. Then, we injected fluorescently modified MVPs or their unconjugated counterparts directly into a variety of solid tumor models in mice. By taking longitudinal in vivo fluorescence measurements of the intratumoral (IT) drug signal over multiple days, we measured the IT half-life of each treatment. Results: Based on binding affinity measurements, we found that MVP potency increased directly with protein valency, and at high valency, the potency of MVPs were substantially greater than the unconjugated protein controls. Multivalent conjugation also increased the hydrodynamic radius of the MVPs to at least ten times larger than the unconjugated therapeutics. This large size was sufficient to slow the diffusion of MVP immunotherapies through dense tissues, such as solid tumors, as demonstrated by our in vivo studies. MVPs exhibited a higher IT drug signal with a more durable gradient within the tumor compared to the unconjugated controls, resulting in an extension of their IT half-lives by >5X in mouse solid tumors. Conclusions: The MVP platform can be used to modulate the potency and therapeutic durability for a wide range of immunotherapy targets. Further, the MVPs stay focused within the tumor after IT injection where they could generate a sustained anti-tumor immune response with minimal systemic exposure. Therefore, we expect MVP immunotherapies to have a better safety profile than IT or systemic delivery of an unconjugated therapeutic. We will continue to develop our internal MVP pipeline to finalize a candidate for IND-enabling studies. We are also seeking collaborations for co-development of additional immunotherapies that could benefit from the extended IT exposure and potency modulation enabled by the MVP platform. Citation Format: Livia Brier, Amy A. Twite, Adam Barnebey, Mavish Mahomed, Wesley M. Jackson. Using a multivalent immunotherapy platform to extend intratumoral therapeutic durability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4157.

Abstract 6225: Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models

Abstract Chimeric antigen receptor (CAR) - engineered natural killer (NK) cell therapy has emerged as a promising platform for adoptive immunotherapy for cancer. However, CAR-NK or CAR-T therapy so far has achieved limited efficacy in solid tumors compared with hematologic malignancies. One of the challenges is the lack of prevalent tumor-specific surface antigen target in solid tumors. Hence, we are developing a novel oncolytic vaccinia virus (VV) to deliver a dual functional glycol-immune checkpoint inhibitor and universal tumor cell marker combined with CAR-NK cell therapy to increase anti-tumor efficacy. Using the vaccinia virus deleted in both viral TK and VGF genes (vvDD) to drive the expression of a membrane-bound sialidase derived from actinomyces viscosus (avSial) under the viral late promoter, we observed efficient desialylation of both VV-infected and non-infected tumor cells, which would alleviate the suppression of sialic acid on NK and other immune cells within TME. Further, the surface bound avSial on VV-infected tumor cells can also serve as a universal target for avSial-CAR NK cells, with less concern for cross-reactivity to normal human tissues and antigen loss. We developed anti-avSial antibodies through mice immunization study. The selected single chain variable fragments (scFV) were constructed in CAR format containing CD28 CD3 ζ for co-stimulation and human IL-15 gene for better NK persistence and function. The binding affinity of anti-avSial scFv to avSialidase were further screened in CAR constructs-transfected 293T cells. The selected anti-avSial scFv CAR were packaged into gamma retroviral vectors to transduce activated and expanded NK cells derived from healthy donor peripheral blood. For proof-of-concept, target tumor cell lines A375, A549 and HT-29 expressing transmembrane sialidase and GFP were also generated. Compared with CD19 CAR NK and none transduced (NT) NK cells, avSial CAR NK had markedly increased cytotoxicity against avSial-expressing tumor cells in co-culture assays at low E:T ratios <= 1:2. Upon tumor cell rechallenge, avSial CAR NK also controlled the tumor growth significantly better than CD19 CAR NK or NT NK cells. Furthermore, avSial CAR NK completely eliminated A375 tumor spheroids expressing transmembrane sialidase whereas CD19 CAR NK or NT NK cells only transiently controlled the tumor spheroids growth. Taken together, we have developed an effective avSial targeting CAR IL15 NK that will be used to demonstrate enhanced efficacy and versatility in combination with avSial-armed VV for treatment of solid tumors. Citation Format: Xiaomei Wang, Guowei Wei, Keshav B. Karki, Winnie Chan, Mariya Viskovska, Andrew Williams, Nancy Chang, Haiyan Jiang. Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6225.

Abstract 4202: Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy

Abstract High-grade gliomas are the most aggressive and refractory of brain tumors. Historical approaches to treating glioblastoma (GBM) using chimeric antigen receptor modified T cells (CAR-T) have been challenging, without a lasting benefit to patients, often due to tumor heterogeneity and a lack of tumor specific antigens. We report preliminary in vitro design and optimization of a γδ T cell-based CAR-T using Chlorotoxin (CLTX-CAR), a peptide isolated from the venom of the Israeli Deathstalker Scorpion that efficiently binds to GBM and other tumor cells with limited cross reactivity to normal tissues. This CLTX-CAR (CTLX-CAR-noZ) omits a CD3z signaling domain to maintain the natural innate targeting and tumor recognition properties of the γδ T cell through its endogenous T cell and NKG2D receptors, and CD16-based antibody-dependent cellular cytotoxicity (ADCC). This construct also includes our Drug Resistant Immunotherapy (DRI) platform that incorporates an additional p140K-MGMT encoding gene into the construct. This confers resistance to alkylating agents such as temozolomide (TMZ) that allows our DRI γδ T cells to remain viable and functional when combined with chemotherapy to force high-expression of natural killer group D ligands (NKG2DL) on the surface of tumor cells. As expected, the non-signaling, CLTX-CAR-noZ modified Jurkat cells did not result in T cell activation, as compared to the highly activated CD69 corresponding to signaling CLTX-CAR-CD3z transduced Jurkat cells. Conversely, CTLX-CAR-noZ transduced γδ T cells demonstrated higher cytotoxicity against U87MG and U251MG GBM cell lines over unmodified control γδ T cells. Dual-CLTX-CAR constructs lacking CD3z and using 2x CLTX peptides as the targeting domain (dCLTX-CAR-noZ) exhibited up to 90% lentiviral transduction efficiency into γδ T cells. When co-cultured in suspension with GBM cells in vitro, dCLTX-CAR-noZ γδ T cells demonstrated an average cytotoxicity of 62.1% against U87MG-GFP cells at an E:T ratio of 4:1 in comparison to 35.4% with non-transduced γδ T control cells. Time-lapsed microscopic recording over a period of 15hrs also verified killing of U251MG-GFP cells by dCLTX-CAR-noZ γδ T cells when cocultured with GBM cells at an E:T ratio of 2:1. Serial-killing of tumor cells by γδ T cells was documented. We are advancing this approach to xenograft animal models and as a clinical development candidate (INB-300) for both GBM and other extracranial tumors. Citation Format: Lei Ding, Yanjie Li, Evan Klumpp, Lawrence Lamb. Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4202.

Abstract 568: A novel memory-like NK cell CAR targeting proximal mesothelin domain shows promising preclinical activity in ovarian cancer using cell lines and patient derived organoids

Abstract Epithelial ovarian cancer (OVC) is the leading cause of death among gynecological malignancies. Natural killer (NK) cells are an attractive platform for immunotherapy owing to their superior safety, multiple mechanisms of target killing, and reduced risk of alloreactivity. Recently, paradigm-shifting studies have shown that brief activation of NK cells with inflammatory cytokines induces differentiation into cytokine induced memory-like (CIML) NK cells. CIML NK cells have demonstrated potent anti-leukemia activity in preclinical settings and in recent clinical trials and therefore represent a novel platform for genetically modified chimeric antigen receptor (CAR) cells for OVC therapy. Mesothelin (MSLN) is expressed in around 82% of OVCs and its expression is corelated with chemoresistance, increased metastasis, and poor overall survival in patients. We hypothesize arming CIML NK cells with a CAR targeting the proximal mesothelin domain will enhance their antitumor responses. We designed and tested anti-MSLN CAR CIML NK cells for their anti-cancer functionality against OVC cell lines and patient-derived organoids (PDOs). We first validated the mesothelin expression in 13 OVC patient samples which, based on pathological scoring showed high mesothelin in 8, moderate in 3, and low in 2 patient samples. PDOs also showed high expression of mesothelin, confirming it as an attractive candidate for CAR cells. We designed the aMSLN-CAR using an ScFv sequence against the proximal domain of the protein with 4-1BB and CD3ζ as co-stimulatory domains. Primary NK cells were purified from peripheral blood, activated using IL-12, IL-18, and IL-15 to afford CIML NK cells. The CAR gene was transduced into CIML NK cells via our optimized baboon lentiviral system to achieve a high transduction efficiency of 40-60%. The direct cytotoxic effect of these NK cells was tested against OVC cell lines. OVC cell lines were co-cultured with NK cells at various effector:target (E:T) ratios for 6 hours and analyzed using apoptotic markers. Compared to CIML NK cells, aMSLN-CAR CIML NK cells showed enhanced cytotoxicity against OVCAR3 (47.9% vs 14.8%), SKOV3 (29.3% vs 8.0%) and OVCAR8 (60.7% vs 39.8%) at 10:1 ratio. To test the therapeutic potential of CAR CIML NK cells in vitro, three different PDOs were used. Cytotoxicity was determined by evaluating apoptotic EpCAM+ cancer cells amongst the organoid cell population after 18-hour co-culture with NK cells. The aMSLN-CAR CIML NK cells were shown to cause increased apoptosis in cells from PDOs compared to CIML NK cells; 58.2% vs 11.0%, 4.5% vs 61.0%, and 10.5% vs 32.0%. We are currently evaluating the in vivo functionality of aMSLN-CAR CIML NK cells in a xenograft mouse model using OVC cell lines and PDOs. The successful application of CAR CIML-NK cells will pave way for the use of NK cell platforms for other solid tumors. Citation Format: Mubin Tarannum, Juliana A. Vergara, Yasmin Abdulhamid, Khanhlinh Dinh, Katherine N. Lynch, Sarah Hill, Rizwan Romee. A novel memory-like NK cell CAR targeting proximal mesothelin domain shows promising preclinical activity in ovarian cancer using cell lines and patient derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 568.

Abstract 5617: A novel genetically modified oncolytic vaccinia virus selectively replicates in tumors to activate an inflammatory TME and induce tumor regression

Abstract Oncolytic viruses (OVs) have been widely investigated as cancer immunotherapy platforms and are represented by a diverse group of DNA and RNA viruses. We are currently investigating a genetically modified oncolytic vaccinia virus as a best-in-class tumor-selective investigational product for the treatment of solid tumors. Specifically, the viral thymidine kinase (TK) has been deleted to enhance safety by greatly limiting replication in healthy tissues while taking advantage of relatively higher endogenous TK levels in tumors. In addition to tumor-selective replication, the oncolytic vaccinia virus encodes an IL-2 variant (IL-2v) protein, which diminishes preferential activation of immunosuppressive Treg cells while preserving activation of CD8+ and NK cells and avoiding toxicities associated with wild type IL-2. Using a murine surrogate of the oncolytic vaccinia virus, we assessed preclinical efficacy in multiple murine syngeneic tumor models demonstrating selective tumor localized replication resulting in tumor growth inhibition. Consistent with tumor selective virus replication, viral copy number and IL-2v transgene levels were increased and had prolonged expression in the tumors compared to the periphery. Intratumoral colocalization of the virus and IL-2v was confirmed by IHC and ISH, respectively. Tumor growth inhibition was associated with increases in tumor infiltrating CD8 and NK effector cells. Additionally, efficacy of the mouse surrogate virus was associated with systemic increases in circulating total CD8 and NK cells as well as pSTAT5+ CD8 and NK cells in the blood. In summary, we have demonstrated that arming a TK-deficient oncolytic vaccinia virus with an IL-2v transgene can effectively couple short-term destruction of tumor cells through oncolysis with the activation of anti-tumor immune response in the tumor microenvironment. These data support the study of this therapy in Phase 1 clinical trials. Citation Format: Anne-Laure Goenaga, I-Ming Wang, Bryan S. Clay, Susanne Lang, Annabel Wang, Peter Weady, Smitha P.S. Pillai, Sripad Ram, Leah Mitchell, Michael Eisenbraun, Joseph Binder, Liliana Maruri Avidal, David Kirn, Robert Hollingsworth, Clare Lees. A novel genetically modified oncolytic vaccinia virus selectively replicates in tumors to activate an inflammatory TME and induce tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5617.

Current Status and Future Directions of Bacteria-Based Immunotherapy.

With the in-depth understanding of the anti-cancer immunity, immunotherapy has become a promising cancer treatment after surgery, radiotherapy, and chemotherapy. As natural immunogenicity substances, some bacteria can preferentially colonize and proliferate inside tumor tissues to interact with the host and exert anti-tumor effect. However, further research is hampered by the infection-associated toxicity and their unpredictable behaviors in vivo. Due to modern advances in genetic engineering, synthetic biology, and material science, modifying bacteria to minimize the toxicity and constructing a bacteria-based immunotherapy platform has become a hotspot in recent research. This review will cover the inherent advantages of unedited bacteria, highlight how bacteria can be engineered to provide greater tumor-targeting properties, enhanced immune-modulation effect, and improved safety. Successful applications of engineered bacteria in cancer immunotherapy or as part of the combination therapy are discussed as well as the bacteria based immunotherapy in different cancer types. In the end, we highlight the future directions and potential opportunities of this emerging field.

Allogeneic gamma delta T cells as adoptive cellular therapy for hematologic malignancies.

Cancer immunotherapy, especially T-cell driven targeting, has significantly evolved and improved over the past decade, paving the way to treat previously refractory cancers. Hematologic malignancies, given their direct tumor accessibility and less immunosuppressive microenvironment compared to solid tumors, are better suited to be targeted by cellular immunotherapies. Gamma delta (γδ) T cells, with their unique attributes spanning the entirety of the immune system, make a tantalizing therapeutic platform for cancer immunotherapy. Their inherent anti-tumor properties, ability to act like antigen-presenting cells, and the advantage of having no major histocompatibility complex (MHC) restrictions, allow for greater flexibility in their utility to target tumors, compared to their αβ T cell counterpart. Their MHC-independent anti-tumor activity, coupled with their ability to be easily expanded from peripheral blood, enhance their potential to be used as an allogeneic product. In this review, the potential of utilizing γδ T cells to target hematologic malignancies is described, with a specific focus on their applicability as an allogeneic adoptive cellular therapy product.

A biomarker-guided Bayesian response-adaptive phase II trial for metastatic melanoma: The Personalized Immunotherapy Platform (PIP) trial design.

TPS9599 Background: Anti-PD1-based immunotherapies have been approved for many cancer types and are now a standard therapy for advanced melanoma. Despite this, ̃50% of advanced melanoma patients (pts) fail to respond or eventually progress after response. It is therefore critical to identify pts with a low likelihood of response to anti-PD1-based therapy and efficiently assess activity of rationally-selected alternative novel immunotherapies. Methods: We designed this investigator-initiated phase II PIP-Trial to evaluate two consecutive biomarker testing platforms, followed by the activity of rationally selected 5 novel agents in pts with advanced melanoma. Two separate pt populations are included: Part-A) treatment-naïve pts predicted to be resistant to either anti-PD-1 alone or combined with ipilimumab using Biomarker Test-1); and Part-B) pts who had progressed on 1 prior line of PD1-based therapy. Part-A) is a Bayesian adaptive multi-arm multi-stage design using response adaptive randomisation after a burn-in period where pts are randomised to the existing arms with equal probability. From then on, regular interim analyses will be carried out with the objective to either drop poorly performing arms or continue. Part-B) is an open platform without control that combined a selection and an expansion phase to identify best novel agent(s) as second-line therapy. Expansion phase decisions will be based on enrichment for biomarker Test-2. Dropping an arm occurs when the posterior probability of observing a clinically significant effect on the primary outcome (i.e. 6-month RECIST objective response rate (ORR)) is low. The operational characteristics of the design were investigated through simulations considering 4 plausible scenarios with 40% ORR in the control arm (anti-PD1 + Anti-CTLA4). Simulations were based on the upcoming R package BATS. Part-A has at least 85% power to detect a 30% absolute improvement in ORR with respect to the control arm (with a max N = 216 – Table below). Part-B will be able to select two promising treatments in the expansion phase and formally test their efficacy against a minimum ORR of 25% at 80% power (max N = 150). [Table: see text]

Cancer immunotherapy based on image-guided STING activation by nucleotide nanocomplex-decorated ultrasound microbubbles.

The cytosolic innate immune sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is crucial for priming adaptive antitumour immunity through antigen-presenting cells (APCs). Natural agonists, such as cyclic dinucleotides (CDNs), activate the cGAS-STING pathway, but their clinical translation is impeded by poor cytosolic entry and serum stability, low specificity and rapid tissue clearance. Here we developed an ultrasound (US)-guided cancer immunotherapy platform using nanocomplexes composed of 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) electrostatically bound to biocompatible branched cationic biopolymers that are conjugated onto APC-targeting microbubbles (MBs). The nanocomplex-conjugated MBs engaged with APCs and efficiently delivered cGAMP into the cytosol via sonoporation, resulting in activation of cGAS-STING and downstream proinflammatory pathways that efficiently prime antigen-specific T cells. This bridging of innate and adaptive immunity inhibited tumour growth in both localized and metastatic murine cancer models. Our findings demonstrate that targeted local activation of STING in APCs under spatiotemporal US stimulation results in systemic antitumour immunity and improves the therapeutic efficacy of checkpoint blockade, thus paving the way towards novel image-guided strategies for targeted immunotherapy of cancer.

Adoptive Immunotherapy With Engineered iNKT Cells to Target Cancer Cells and the Suppressive Microenvironment.

Invariant Natural Killer T (iNKT) cells are T lymphocytes expressing a conserved semi-invariant TCR specific for lipid antigens (Ags) restricted for the monomorphic MHC class I-related molecule CD1d. iNKT cells infiltrate mouse and human tumors and play an important role in the immune surveillance against solid and hematological malignancies. Because of unique functional features, they are attractive platforms for adoptive cells immunotherapy of cancer compared to conventional T cells. iNKT cells can directly kill CD1d-expressing cancer cells, but also restrict immunosuppressive myelomonocytic populations in the tumor microenvironment (TME) via CD1d-cognate recognition, promoting anti-tumor responses irrespective of the CD1d expression by cancer cells. Moreover, iNKT cells can be adoptively transferred across MHC barriers without risk of alloreaction because CD1d molecules are identical in all individuals, in addition to their ability to suppress graft vs. host disease (GvHD) without impairing the anti-tumor responses. Within this functional framework, iNKT cells are successfully engineered to acquire a second antigen-specificity by expressing recombinant TCRs or Chimeric Antigen Receptor (CAR) specific for tumor-associated antigens, enabling the direct targeting of antigen-expressing cancer cells, while maintaining their CD1d-dependent functions. These new evidences support the exploitation of iNKT cells for donor unrestricted, and possibly off the shelf, adoptive cell therapies enabling the concurrent targeting of cancer cells and suppressive microenvironment.

Targeted Cancer Immunotherapy by Nanoparticle T Cell Engagers

IntroductionAcute myeloid leukemia (AML) is the most common type of leukemia. Emerging immunotherapies such as chimeric antigen receptor T (CAR‐T) cells and T cell engagers (TCEs), have the potential to enhance T cell recognition and response against cancers, but come with serious limitations such as short half‐life, complex production, high cost, and inability to customize for another antigen. To overcome these limitations, we previously developed a nanoparticle‐based T cell engagers (nanoTCEs) technology that has a longer half‐life (60h vs 2h for TCEs), is convenient to make, easily customizable, and robustly activates T cells for cancer killing. In this study, we hypothesized that nanoTCEs targeting CD33/CD3 would effectively activate T cell response in an AML disease model.Material and MethodsLiposomes were produced via the thin‐film hydration method with cholesterol, DPPC, and DSPE‐PEG200 at a molar ratio of 30:65:5 respectively (Figure 1A). CD33/CD3 nanoTCEs were created by conjugating anti‐CD3 and anti‐CD33 monoclonal antibodies (mAbs) on liposome surface with streptavidin‐biotin chemistry. Liposome binding to human T cells and AML cell lines was performed with fluorescently labeled nanoTCEs for 2 hours. The activation of T cell subsets and killing of AML cell lines was analyzed by flow cytometry after 4 days in a 3D culture model. For in vivo efficacy, immunodeficient NCG mice were inoculated with AML cell line and received Isotype/CD3 or CD33/CD3 nanoTCEs injections (0.5 mg/mouse) weekly and monitored for tumor progression and survival.ResultsAML cell lines (K052, MOLM‐14. NOMO‐1, and THP‐1) exhibited high expression of CD33 compared to controls. CD33/CD3 nanoTCEs bound to human T cells and AML cell lines with higher specificity than isotype controls. In addition, the CD33/CD3 nanoTCEs induced increased activation of both CD4+ and CD8+ T cells (Figure 1B, 1C) and achieved greater AML killing compared to the untreated and Isotype/CD3 groups (Figure 1D). Lastly, our in vivo results showed that weekly CD33/CD3 nanoTCE injections extended survival and decreased tumor progression of AML bearing mice compared to Isotype/CD3 nanoTCEs treated mice. By day 66 of the in vivo study, none of the Isotype/CD3 cohort was alive, while 100% of the CD33/CD3 cohort remained living (Figure 2).Discussion and ConclusionThis study shows that nanoTCEs are a promising novel platform for immunotherapy in AML. CD33/CD3 nanoTCEs demonstrated specific engagement of T cells to AML cells, which induced T cell activation and AML cell killing both in vitro and in vivo. It not only circumvented the limitations of traditional T cell engagers, but also presented numerous advantages such as short production time, low cost, and superior pharmacokinetic profile. Moreover, this technology is highly customizable and can be tailored to target multiple cancer antigens simultaneously. The current findings provide a strong preclinical basis for future first‐in‐human clinical trials of CD33/CD3 nanoTCEs as a novel AML immunotherapy.